Bowles Center for Alcohol Studies, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA. darin_knapp@med.unc.edu

Abstract

Stress has been shown to facilitate ethanol withdrawal-induced anxiety. Defining neurobiological mechanisms through which stress has such actions is important given the associated risk of relapse. While CRF has long been implicated in the action of stress, current results show that stress elevates the cytokine TNFα in the rat brain and thereby implicates cytokines in stress effects. In support of this view, prior TNFα microinjection into the central amygdala (CeA) of rats facilitated ethanol withdrawal-induced anxiety-a response that could not be attributed to an increase in plasma corticosterone. To test for a possible interaction between cytokines and CRF, a CRF1-receptor antagonist (SSR125543) administered prior to the repeated administration of TNFα or MCP-1/CCL2 reduced the magnitude of the withdrawal-induced anxiety. This finding provided evidence for cytokine action being dependent upon CRF. Additionally, the sensitizing effect of stress on withdrawal-induced anxiety was reduced by treating the repeated stress exposure prior to ethanol with the MEK inhibitor SL327. Consistent with cytokines having a neuromediator function distinct from a neuroimmune action, TNFα increased firing rate and GABA release from CeA neurons. Thus, an interaction of glial and neuronal function is proposed to contribute to the interaction of stress and chronic ethanol. Interrupting this potential glial-neuronal interaction could provide a novel means by which to alter the development of emotional states induced by stress that predict relapse in the alcoholic.

A CRF-1R antagonist blocks the sensitizing effect of cytokines (MCP-1 or TNFα; 100 ng ICV) on ethanol withdrawal anxiety. Rats were administered SSR125543 prior to either cytokine during each of the first two of three ethanol withdrawal periods. Behavior was recorded during the third withdrawal. CD = control diet; ED = ethanol diet, *p<0.05 vs CD/Veh; +<0.05 vs ED-TNFα or ED-MCP-1.

Physiological responses of amygdala neurons to TNFα. Representative neuronal firing rates in a neuron from an amygdala slice preparation before, during and after TNFα (1.25 nM) exposure (upper left panel). The firing rate increase relative to the average of the pre and post-TNFα periods was significantly greater than rates obtained from non-TNFα exposed cells. A similar comparison of the effect of TNFα (1.25 nM) on GABA release in the amygdala slice is shown in the lower right panel lower right panel). Representative traces of mIPSCs are shown before, during, and after TFNα in the upper right panel. Numbers in parentheses are number of cells. mIPSCs = mini inhibitory post-synaptic currents. *p < 0.05 versus control.

This stress-facilitated effect on withdrawal anxiety is sensitive to pharmacological manipulation (). Among the drugs that prevented this sensitization of withdrawal anxiety was a CRF1R antagonist. The CRF1R antagonist SSR125543 was administered prior to ICV administration of TNFα and MCP-1 that were given prior to the chronic ethanol exposure to consequences on ethanol withdrawal anxiety. The ICV cytokines administered twice prior to the subthreshold chronic ethanol experience produce social interaction deficits similar to those previously reported (). The CRF1R antagonist blocked the cytokine-induced sensitization of withdrawal anxiety. Given that the CRF1R antagonist prevents sensitization by stress as well as by CRF (; ), our new results are consistent with CRF impacting cytokines (; ; ). A schematic representation of cytokine/CRF interactions that may be involved in this response is depicted in . This potential involvement of CRF in microglia actions is based in part on upon , ) who demonstrated that CRF activation of CRF-1Rs on microglia facilitate release of cytokines. These and other results suggest the possibility that stress-induced release of CRF can release cytokines (, ), which in turn facilitate further CRF release (). The results described herein extend these observations by implicating an interaction of cytokines and CRF in stress associated with alcohol withdrawal and by implicating CRF in sensitized withdrawal anxiety following repeated cytokine exposures (). Overall, these experiments are consistent with a growing body of evidence implicating CRF in stress and chronic ethanol effects that influence emotional behavior and ethanol consumption (Breese et al., 2010; for reviews).